The human lung is composed of a large number of small sacs or alveoli in which gases are exchanged between the blood and the air spaces of the lung. In healthy individuals, this exchange is mediated by the presence of a protein-containing surfactant complex which is synthesized in the microsomal membranes of type II alveolar cells. In the absence of adequate levels of this complex, a lung cannot properly function--i.e., the alveoli collapse during exhalation, and cannot be subsequently re-inflated by inhaling. Thus, the untreated inability to synthesize this complex may result in death or in severe physical damage if left untreated.
The best documented instance of inadequate surfactant complex levels occurs in premature infants and infants born after complicated pregnancies, and is widely known as respiratory distress syndrome (RDS). A widely publicized form of this syndrome has been designated hyaline membrane disease, or idiopathic RDS. RDS is currently the leading cause of infant mortality and morbidity in the United States and in other developed countries, and substantial efforts have been directed to diagnosis and treatment. Current treatment has focused on mechanical (pressure) ventilation which, at best, is an invasive stop-gap measure that often results in damage to the lung and other deleterious side effects, including complications such as bronchopulmonary dysplasia, interstitial emphysema and pneumothorax. Mental retardation has also resulted on occasion when this treatment was used (Hallman, M., et al, Pediatric Clinics of North America (1982) 29:1057-1075).
Limited attempts have been made to treat the syndrome by surfactant substitution. This would be a method of choice, as, in general, only one administration is required, and the potential for damage is reduced. For example, Fujiwara, et al, Lancet (1980) 1:55-used a protein-depleted surfactant preparation derived from bovine lungs, while Hallman, M., et al, Pediatrics (1983) 71:473-482 used a surfactant isolated from human amniotic fluid to treat a limited number of infants with some success U.S. Pat. No. 4,312,860 to Clements discloses an artificial surfactant which contains no protein and is said to be useful in this approach although no data are shown. In short, surfactant substitution has not been widely used clinically.
The preferred surfactant substitute would be the lung surfactant complex itself. This complex is composed of apoprotein, two phospholipids (dipalmitoyl phosphocholine (DPPC) and phosphatidyl-glycerol (PG)) which are present in major amount, several lipid components present in only very minor amount, and calcium ions. The apoprotein contains proteins having molecular weights of the order of 32,000 daltons and very hydrophobic proteins of the order of about 10,000 daltons (King, R.J. et al, Am J Physiol (1973) 224:788-795). The 32,000 dalton protein is glycosylated and contains hydroxyproline.
A major reason for the limited progress in surfactant replacement therapy has been the lack of availability of the protein portion of the complex. Replacement therapies have focused on attempts to use the lipid components alone, and it appears that the performance of such treatment can be markedly improved by addition of the apoprotein (Hallman, M., et al, Pediatric Clinics of North America (1982) (supra)). At present, however, these proteins are available only from normal adult human lung, and from amniotic fluid. Even efficient isolation procedures would not provide an adequate supply. Thus, it would be desirable to have available a method for producing practical quantities of apoprotein for use alone or in conjunction with the saturated phospholipid portion of the complex.
Related PCT patent application WO86/03408 describes the recombinant production of the human ASP protein of about 32 kd, the retrieval of DNA sequences encoding various canine ASP proteins and the retrieval of a single representative of the human ASP protein group of about 10 kd molecular weight. It is now clear that efficient production of the "10K" group is required for use in adequate therapy.
The additional related PCT patent application WO87/06588, published Nov. 5, 1987, gives further description of these 10K proteins and their encoding DNAs. FIGS. 1 and 2 of that application show the full-length cDNAs encoding precursors of canine and human SP-18-derived protein. The mature human protein is described to begin at the phenylalanine residue encoded at codon 201 of the full-length sequence. The construction of vectors for expression of the SP-18 precursor in both mammalian and bacterial cells is described in detail. Expression of the full-length precursor in mammalian cells yielded 43 kd and 25 kd precursor proteins as determined on SDS-PAGE. The 25 kd product is stated to be the glycosylated form of a 181 amino acid sequence spanning Phe-201 Glu-381 encoded in this sequence. Certain modified forms of the human protein to provide cleavage sites which may be helpful in providing more uniform production of mature forms of the precursor are also described. Bacterial expression of the SP-18 cDNA is also described.
FIGS. 5 and 6 of PCT application WO87/06588 show the DNA and deduced amino acid sequences of two cDNA clones encoding the precursors for the smaller molecular weight 5 kd-8 kd proteins, designated SP-5. Like the SP-18 cDNA, these clones are disclosed to encode a precursor for the smaller 5 kd-8 kd proteins isolated. The putative N-terminus is stated to be Phe or Gly at codons 24 and 25 of this sequence; it is postulated that the mature C-terminus of these proteins is at Gln-108 for the 8 kd protein and Glu-80 or Thr-65 for the 5 kd protein. Expression of this cDNA in mammalian and bacterial cells is also described.
The disclosures of the two above-cited PCT applications, WO86/03408 and WO87/06588, are incorporated herein by reference.
The present application describes various SP-5-related peptides which are effective as lung surfactant proteins. These SP-5 analogs and fragments can be prepared by chemical synthesis or by recombinant methods and offer specific members of the repertoire of lung surfactant proteins useful in treatment of respiratory diseases and symptomologies.
The parent application hereto, U.S. application Ser. No. 07/117,009, also describes the 10K group of proteins in some detail. The disclosure of that application is hereby incorporated by reference in its entirety, and reference may be had thereto for material not explicitly described or explained herein. The present application is based on further studies of the human SP-5 protein, and in particular is directed to analogs of that protein which have now been found to have ASP activity. The analogs presently described and claimed, in addition to retaining the stability and biological activity of the native polypeptide, are less susceptible to aggregation than native 5 kd protein.